SIRT6 Regulates TGF-β Signaling Pathway In Cardiac Fibrosis Induced By AngⅡ

BackgroundIn recent years,the incidence rate of cardiovascular diseases such as hypertension and coronary heart disease has been increasing.These diseases eventually develop into heart failure,which seriously threatens human life and health.Cardiac fibrosis（CF）is a major process in cardiovascular diseases,which can cause contraction and/or diastolic dysfunction of the heart and eventually lead to heart failure.Studies have shown that activation of the renin-angiotensin system（RAS）is a key factor in the progression of heart failure,Angiotensin II（Ang II）,as the main biological peptide of RAS,plays an important role in myocardial interstitial fibrosis by enhancing the activation of connective tissue growth factor（CTGF）-chemokine FKN signal,leading to myocardial hypertrophy,cardiac dysfunction,and aggravation of heart injury.Sirtuin 6（SIRT6）is a conservative nicotinamide adenine dinucleotide-dependent deacetylase that has been shown to play important roles in regulating heart function,energy metabolism,and aging.Studies have shown that SIRT6 expression is significantly reduced in the heart of patients with heart failure.At present,drug development targeting SIRT6 has attracted widespread attention.The conversion of cardiac fibroblasts into myofibroblasts is the initial event of cardiac remodeling.Transforming growth factor-β（TGF-β）is currently the strongest and most widely studied fibrosis factor known to participate in the pathogenesis of many cardiovascular diseases,including hypertension、vascular restenosis、atherosclerosis,cardiac hypertrophy and heart failure.TGF-βexerts a pleiotropic effect on cardiomyocytes and regulating cell growth,fibrosis and inflammation.As an important extracellular matrix regulator,TGF-β-mediated vascular fibrosis involves a variety of mechanisms,including the Smad signaling pathway and the activation of protein kinase pathways.TGF-β1 has the strongest ability to induce cell and tissue fibrosis.Under pathological and physiological conditions,fibroblasts can synthesize and secrete TGF-β1,promote the secretion of diseased extracellular matrix,and increase the degree of fibrosis.When TGF-β1 exists as a protein complex,it does not express biological effects.After activation by hydrolase,fibroblasts can be induced to differentiate into myofibroblasts.Classical TGF-β1 signaling pathways include TGF-β1 binding to cell membrane surface receptors,intracellular Smad_2/3phosphorylation,binding to Smad₄ to form complexes,and complexes activating gene expression in the nucleus.Therefore,We make the following scientific assumptions:（1）Does SIRT6 have a protective effect on AngⅡ-induced cardiac fibrosis?（2）Is the function of SIRT6 reverse-regulating cardiac fibroblasts into myofibroblasts related to inhibition of the TGF-β1/Smad pathway?ObjectivesObserve the improvement of heart function and myocardial interstitial fibrosis in AngⅡ-induced mouse cardiac fibrosis model after NMN increasing the expression of SIRT6 in mouse heart,and detect the changes of cardiac fibrosis related proteins.Further explore whether SIRT6 can improve cardiac fibrosis in mice by inhibiting Smad_2/3 phosphorylation to reversely regulate the TGF-β1/SMAD signaling pathway.Methods（1）To construct the mouse.model of cardiac fibrosis,the Alzet1004 micropump was implanted in the back of the mouse,and 0.726mg/ml of AngⅡsolution was pumped into the mouse at a constant rate of 0.11μl/h,which is equivalent to continuous administration of AngⅡat a rate of 750ng/kg·min.On this basis,intraperitoneal injection of NMN increased SIRT6 expression in mouse heart.The experiment mice were divided into four groups:control group（CON group）,intraperitoneal injection of NMN group（NMN group）,cardiac fibrosis model group（AngⅡgroup）,cardiac fibrosis model+intraperitoneal injection of NMN group（AngⅡ+NMN group）.After 4 weeks of continuous pumping,the left ventricular end-diastolic volume（LVEDV）and left ventricular end-systolic volume（LVESV）were measured by a small animal ultrasound system,then we can get the left ventricular ejection fraction（LVEF）and the left ventricular fractional shortening（LVFS）.Collect the serum of the mice,and then measure the Atrial natriuretic peptide（ANP）and Brain natriuretic peptide（BNP）by enzyme-linked immunosorbent assay（ELISA）.The heart of the mouse was taken and embedded in paraffin,and then the degree of myocardial interstitial fibrosis was detected by HE staining,Masson staining and Sirius scar staining.Mouse heart proteins were extracted and the expression levels of collagenⅠ,CollagenⅢ,α-SMA and Sirt6 in each group were detected by Western blot,and gene expressions were verified by RT-PCR.（2）Primary cultured neonatal mouse myocardial fibroblasts were passed to the third passage,and then the P3 fibroblasts were treated with 0n M,25n M,50n M,100n M,200n M,400n M,800n M concentrations of AngⅡ.CCK-8 experiments were performed 48 hours later to determine the experimental concentration of AngⅡby observing the cell proliferations.Ad-Zs Green-sh RNA-m Sirt6 adenovirus was constructed.Ad-Sirt6 sh RNA was used to transfect P₃ fibroblasts.The transfection was observed by fluorescence microscopy,Western blot was used to verify the transfection efficiency of the virus.Cell experiments were divided into four groups:P3 cardiac fibroblasts cultured in normal medium（CON group）,P3 cardiac fibroblasts transfected with Ad-Sirt6 sh RNA,and cultured in normal medium（Ad-Sirt6 sh RNA group）,P3 cardiac fibroblasts treated with100n M AngⅡ（AngⅡgroup）and P3 cardiac fibroblasts transfected with Ad-Sirt6 sh RNA,and then treated with 100n M AngⅡ（AngⅡ+Ad-Sirt6 sh RNA group）.48 hours later,four groups of cells were subjected toα-SMA immunofluorescence staining,and the degree of differentiation of fibroblasts to myofibroblasts was determined according to the red fluorescence.Western blot was used to detect the expression ofα-SMA,SMAD_2/3,and p-SMAD_2/3,and the expression of fibrosis-related genes,such as CollagenⅠ,CollagenⅢ,α-SMA and p-SMAD_2/3 were detected by RT-PCR.Results（1）Cardiac function declines in mice with cardiac fibrosis.Intraperitoneal injection of NMN can up-regulate SIRT6 expression in mouse hearts,improve cardiac function,reduce myocardial interstitial fibrosis.The expressions of cardiac fibrosis-related proteins,such as CollagenⅠ,CollagenⅢ,andα-SMA were all down-regulated（p﹤0.05）.（2）Ad-Sirt6 sh RNA down-regulates the expression of SIRT6 in cardiac fibroblasts,which can promote the proliferation of cardiac fibroblasts induced by AngⅡand differentiating into myofibroblasts.SIR6 may negatively regulate TGF-β/SMAD Signaling pathway to inhibit the proliferation and differentiation of cardiac fibroblasts and reduce cardiac fibrosis caused by AngⅡ.Conclusion1.By up-regulating the expression of SIRT6 in cardiac tissue,it can reduce cardiac fibrosis caused by AngⅡand improve cardiac function.2.By down-regulating the expression of SIRT6 in cardiac fibroblasts,it can promote the proliferation of cardiac fibroblasts induced by AngⅡand differentiate into myofibroblasts.3.SIRT6 may inhibit the proliferation and differentiation of cardiac fibroblasts through negatively regulating the TGF-β/SMAD signal pathway,and alleviate cardiac fibrosis caused by AngⅡ.